Albumin pharmaceutical composition and preparation method therefor

ABSTRACT

Disclosed herein is an albumin pharmaceutical composition, comprising albumin and at least one amino acid having a relative molecular mass of 145-175. Clinically, the albumin pharmaceutical composition can effectively reduce undesirable responses, such as rash, urticaria, anaphylaxis and possible immune responses in the human body caused by albumin polymers and dimers, thereby further ensuring the safety in clinical medication.

TECHNICAL FIELD

The present invention relates to an albumin pharmaceutical composition,and more particularly relates to an albumin nanoparticle pharmaceuticalcomposition comprising an amino acid having a relative molecular mass of145 to 175 or a salt thereof.

BACKGROUND ART

Albumin has been widely used as a nanoparticle drug delivery system, andis mainly used for the delivery of poor water-soluble drugs such astaxanes, docetaxel and the like. Paclitaxel is insoluble in water, inorder to improve the solubility of paclitaxel and make it suitable forintravenous injection, Bristol-Myers Squibb Company mixed it with asurfactant (e.g., polyethoxylated castor oil) and about 50% anhydrousalcohol (as a carrier of paclitaxel). However, in addition to sideeffects per se, the presence of the surfactant and anhydrous alcoholalso results in serious drawbacks, which mainly is strong anaphylacticreaction.

In order to eliminate the strong anaphylactic reaction, the EuropeanPatent EP-A-0584001 and U.S. Pat. No. 5,641,803 disclosed addition ofother substances to paclitaxel pharmaceutical compositions. However, thesubstances, which are added to prevent the anaphylactic reaction, willintroduce side effects caused by these substances themselves into thepharmaceutical compositions.

Further, as a first commercially available albumin nanoparticleformulation, paclitaxel-albumin nanoparticle Abraxane was approved inthe United States in 2005 and was approved by the CFDA in 2008 to beused for the treatment of metastatic breast cancer. Abraxane hasrecently been approved to be used for the treatment of non-small celllung cancer, pancreatic cancer, and the like, in the United States. Inthe preparation of Abraxane, an albumin derived from human blood isused. At present, researchers from different countries are trying to usehuman serum albumin to develop albumin nanoparticles of other drugs,such as docetaxel.

Generally speaking, the biggest advantage of the Abraxane in clinical isthe elimination of the anaphylactic reaction caused by common paclitaxelinjections due to the addition of auxiliaries like polyethoxylatedcastor oil, thereby greatly improving the safety of the product.However, in clinical applications, some patients are also found certainanaphylactic reactions to Abraxane. The primary cause is that theincrease of albumin dimers and polymers formed during storage, resultingin undesirable responses in human body, such as rash, urticaria,anaphylactic reactions and possible immune responses. The increase ofalbumin dimers and polymers in the formulation may also result in easilyaggregation of the formulation, which may affect the physical stabilityof the formulation (“Effect of caprylate and acetyl-tryptophan onstabilization of human albumin”, Progress in Microbiology andImmunology, 1985).

At present, there is no quite effective method for inhibiting theformation of albumin dimers and polymers in an albumin nanoparticlepharmaceutical composition during storage.

Therefore, an urgent problem to be solved in the field is to develop analbumin nanoparticle composition and pharmaceutical formulation, whichcan effectively control the amounts of albumin dimers and/or polymers inthe albumin pharmaceutical composition during storage, and reduceanaphylactic reaction in clinical applications so as to further ensurethe safety in clinical medication.

CONTENTS OF THE PRESENT INVENTION

It is an object of the present invention to provide a method forimproving the quality of an albumin pharmaceutical composition,comprising adding at least one amino acid having a relative molecularmass of 145-175 or a salt thereof during the preparation of thepharmaceutical composition, and the amino acid may be selected from oneor more of arginine, histidine and lysine, preferably arginine and/orhistidine, more preferably arginine; and the amino acid or the saltthereof inhibits the formation and/or the increase of albumin dimersduring the preparation, storage and/or use of the pharmaceuticalcomposition.

It is another object of the present invention to provide a method forcontrolling the increase of albumin dimers during the preparation,storage and/or use of an albumin pharmaceutical composition, comprisingadding at least one amino acid having a relative molecular mass of145-175 or a salt thereof during the preparation of the pharmaceuticalcomposition, wherein the amino acid may be selected from one or more ofarginine, histidine and lysine, preferably arginine and/or histidine,more preferably arginine.

In some embodiments, in the pharmaceutical composition, the albumindimers are present in an amount of no greater than about 10%, morepreferably, no greater than about 7%, most preferably, no greater thanabout 5%, of the total amount of the albumins.

In some embodiments, the above methods inhibit the formation and/or theincrease of albumin polymers during the preparation, storage, and/or useof the pharmaceutical composition.

Preferably, in the pharmaceutical composition, the albumin polymers arepresent in an amount of no greater than about 6%, more preferably, nogreater than about 4%, most preferably, no greater than about 2%, of thetotal amount of the albumins.

In some embodiments, the amino acid or the salt thereof of the presentinvention is added in an amount that can effectively inhibit theformation and/or the increase of albumin dimers in the composition.

It is another object of the present invention to provide a method forpreparing a pharmaceutical composition, comprising dissolving a taxanecompound in a suitable organic solvent to obtain a solution of thetaxane compound; dissolving or diluting an albumin into an aqueoussolvent to obtain an aqueous solution of the albumin; preparing anaqueous solution of an amino acid having a relative molecular mass of145-175 or a salt thereof by using water for injection; and subjectingthe solution of the taxane compound, the aqueous solution of the albuminand the aqueous solution of the amino acid having a relative molecularmass of 145-175 or the salt thereof to dispersive-mixing under highshear force, then ultrafiltration, sterilization by filtration, andoptional lyophilization.

The method does not comprise a step of adding a lyoprotectant prior tothe lyophilization, wherein the lyoprotectant is selected from one ormore of trehalose, sucrose, maltose, lactose, and glycine.

It is another object of the present invention to provide use of an aminoacid or a salt thereof for inhibiting the formation and/or the increaseof albumin dimers in a pharmaceutical composition for a certain periodof time, wherein the amino acid has a relative molecular mass of145-175, and the amino acid may be selected from one or more ofarginine, histidine and lysine, preferably arginine and/or histidine,more preferably arginine.

In some embodiments, at 20-38° C., preferably 22-30° C., more preferably25° C., for a period of more than 6 months, preferably more than 1 year,more preferably 2 years, in the pharmaceutical composition, the albuminmonomers remain in an amount of above 83% of the total amount of thealbumins, the albumin dimers remain in an amount of below 8% of thetotal amount of the albumins, and the albumin polymers remain in anamount of below 5% of the total amount of the albumins, preferably, thealbumin monomers remain in an amount of above 85% of the total amount ofthe albumins, the albumin dimers remain in an amount of below 5% of thetotal amount of the albumins, and the albumin polymers remain in anamount of below 5% of the total amount of the albumins; and/or,

In some embodiments, at 50-65° C., preferably 58-62° C., more preferably60° C., for a period of more than one week, in the pharmaceuticalcomposition, the albumin monomers remain in an amount of above 80% ofthe total amount of the albumins, the albumin dimers remain in an amountof below 9% of the total amount of the albumins, and the albuminpolymers remain in an amount of below 6% of the total amount of thealbumins; preferably, the albumin monomers remain in an amount of above88% of the total amount of the albumins, the albumin dimers remain in anamount of below 6% of the total amount of the albumins, and the albuminpolymers remain in an amount of below 3% of the total amount of thealbumins, more preferably, the albumin monomers remain in an amount ofabove 90% of the total amount of the albumins, the albumin dimers remainin an amount of below 5% of the total amount of the albumins, and thealbumin polymers remain in an amount of below 2% of the total amount ofthe albumins.

It is another object of the present invention to provide a stablealbumin pharmaceutical composition, comprising an albumin and at leastone amino acid having a relative molecular mass of 145-175 or a saltthereof, and in the pharmaceutical composition, the albumin dimmers arepresent in an amount of no more than about 10%, preferably no more thanabout 7%, and more preferably no more than about 5%, of the total amountof the albumins, in at least one year.

In some embodiments, in the pharmaceutical composition, the albuminpolymers are present in an amount of no more than about 6%, preferablyno more than about 4%, more preferably no more than about 2%, of thetotal amount of the albumins, in at least one year.

In the present invention, the amino acid refers to an amino acid havinga relative molecular mass of 145 to 175, and furthermore, the amino acidof the present invention refers to one or more selected from arginine,histidine and lysine, preferably arginine and/or histidine, morepreferably arginine. The weight ratio of the amino acid or a saltthereof to albumin is from 0.1: 1 to 10:1, more preferably from 0.2:1 to5:1.

In some embodiments, the pharmaceutical composition comprises a taxanecompound as an active ingredient.

In some embodiments, the pharmaceutical composition is an albuminnanoparticle pharmaceutical composition.

In some embodiments, the amino acid or the salt thereof is the solestabilizer in the pharmaceutical composition, and the amino acid may beselected from one or more of arginine, histidine, and lysine, preferablyarginine and/or histidine, more preferably arginine.

In some embodiments, the pharmaceutical composition is a lyophilizedpowder injection.

It is another object of the present invention to provide use of thepharmaceutical composition of the present invention in the manufactureof a medicament for treating a cancer.

In some embodiments, the cancer is selected from one or more of prostatecancer, colon cancer, breast cancer, head and neck cancer, pancreaticcancer, lung cancer, ovarian cancer, multiple myeloma, renal cellcarcinoma, melanoma, liver cancer, gastric cancer, and kidney cancer.

It is another object of the present invention to provide an albuminnanoparticle pharmaceutical composition, comprising a taxane compound,an albumin and at least one amino acid having a relative molecular massof 145-175 or a salt thereof, and the composition comprises albuminmonomers in an amount of no less than about 80% of the total amount ofthe albumins, and/or albumin dimers in an amount of no more than about10% of the total amount of the albumins, and/or albumin polymers in anamount of no more than about 6% of the total amount of the albumins, andthe amino acid may be selected from one or more of arginine, histidineand lysine, preferably arginine and/or histidine, more preferablyarginine.

In some embodiments, the amino acid having a relative molecular mass of145-175 comprised in the albumin nanoparticle pharmaceutical compositionof the present invention refers to one or more of arginine, histidine,and lysine, preferably arginine and/or histidine, more preferablyarginine.

In some embodiments, the taxane compound used in the present inventionis selected from one or more of paclitaxel, docetaxel, cabazitaxel, andderivatives thereof.

In some embodiments, the albumin used in the present invention isselected from one or more of recombinant albumin, animal serum albumin,and human serum albumin, preferably human serum albumin.

It is another object of the present invention to provide apharmaceutical formulation, comprising the albumin nanoparticlepharmaceutical composition, wherein the formulation comprises albuminmonomers in an mount of no less than about 80% of the total amount ofthe albumins, and/or albumin dimers in an amount of no more than about10% of the total amount of the albumins, and/or albumin polymers in anamount of no more than about 6% of the total amount of the albumins.

In some embodiments, the pharmaceutical formulation further comprisesone or more pharmaceutically acceptable carriers and/or adjuvants.

It is another object of the present invention to provide use of thealbumin nanoparticle pharmaceutical composition in the manufacture of amedicament for treating a cancer. In some embodiments, the cancer isselected from one or more of prostate cancer, colon cancer, breastcancer, head and neck cancer, pancreatic cancer, lung cancer, ovariancancer, multiple myeloma, renal cell carcinoma, melanoma, liver cancer,gastric cancer, and kidney cancer.

It is another object of the present invention to provide a method forstabilizing an albumin nanoparticle pharmaceutical compositioncomprising a taxane compound, comprising adding an appropriate amount ofat least one amino acid having a relative molecular mass of 145-175 or asalt thereof, wherein the amino acid particularly refers to one or moreamino acid selected from arginine, histidine, and lysine, preferablyarginine and/or histidine, more preferably arginine. The addition of theamino acid or the salt thereof can reduce albumin dimers and/or polymersor inhibit the increase thereof. In the method of the present invention,the addition of the amino acid or the salt thereof can reduce theformation of albumin dimers and/or polymers, and improve the chemicalstability of albumin nanoparticles, thereby fundamentally solving theproblem of the physically and chemically instability of albuminnanoparticles, reducing the kinds and amount of impurities, reducingtoxicity in vitro and in vivo, and improving safety for patients.

In some embodiments, the method comprises preparing an albuminnanoparticle composition comprising a taxane compound under high shearconditions, wherein an appropriate amount of at least one amino acidhaving a relative molecular mass of 145-175 or a salt thereof is addedduring the preparation, and the amino acid particularly refers to one ormore amino acid selected from arginine, histidine, and lysine,preferably arginine and/or histidine, more preferably arginine.

In some embodiments, the method of the present invention comprises thesteps of: dissolving the taxane compound in a suitable organic solventto obtain the solution of the taxane compound; dissolving or dilutingthe albumin into an aqueous solvent to obtain an aqueous solution of thealbumin; preparing the aqueous solution of the amino acid having arelative molecular mass of 145-175 or the salt thereof by using water(preferably, water for injection), wherein the amino acid particularlyrefers to one or more amino acid selected from arginine, histidine, andlysine, preferably arginine and/or histidine, more preferably arginine;and subjecting the solution of the taxane compound, the aqueous solutionof the albumin and the aqueous solution of the amino acid having arelative molecular mass of 145-175 or the salt thereof todispersive-mixing under high shear force, then ultrafiltration,sterilization by filtration, and optional lyophilization.

It is another object of the present invention to provide use of an aminoacid having a relative molecular mass of 145 to 175 or a salt thereoffor stabilizing an albumin nanoparticle pharmaceutical composition or apharmaceutical formulation comprising the albumin nanoparticlepharmaceutical composition. The use according to the present inventioncomprises inhibiting the formation of albumin dimers and/or polymers inthe albumin nanoparticle pharmaceutical composition or thepharmaceutical formulation comprising the albumin nanoparticlepharmaceutical composition during storage by using an appropriate amountof at least one amino acid having a relative molecular mass of 145-175or a salt thereof. The amino acid particularly refers to one or moreamino acid selected from arginine, histidine, and lysine, preferablyarginine and/or histidine, more preferably arginine.

It is another object of the present invention to provide use of an aminoacid having a relative molecular mass of 145-175 or a salt thereof inthe preparation of an albumin nanoparticle pharmaceutical compositionfor reducing adverse reactions in a subject, wherein the albuminnanoparticle pharmaceutical composition comprises a taxane compound, andthe pharmaceutical composition comprises albumin monomers in an amountof no less than about 80% of the total amount of the albumins, and/oralbumin dimers in an amount of no more than about 10% of the totalamount of the albumins, and/or albumin polymer in an amount of no morethan about 6% of the total amount of the albumins.

The use according to the present invention can improve the chemicalstability of albumin nanoparticles, thereby fundamentally solving theproblem of the physical and chemical instability of albuminnanoparticles, reducing the kinds and amount of impurities, reducingtoxicity in vitro and in vivo, and improving safety for patients.

The albumin nanoparticle composition or formulation of the presentinvention and the albumin nanoparticle composition prepared according tothe preparation method of the present invention, can reduce one or moreadverse reactions including, for example, rash, urticaria, anaphylacticreaction, and immune response.

SPECIFIC MODE FOR CARRYING OUT THE PRESENT INVENTION

In the present invention, the following terms have the followingdefinitions.

As used herein, the term “about” refers to a range of numerical valuemodified by the term ±10%, preferably ±5%.

As used herein, the term “amount” refers to weight percentage.

As used herein, the term “relative molecular mass” refers to the mass ofa molecule relative to 1/12 of a carbon atom, with unit of one.

As used herein, the term “amino acid having a relative molecular mass of145-175” refers to a natural amino acid having a relative molecular massin the range of from 145 to 175, and the amino acid specificallyincludes lysine, glutamine, glutamic acid, methionine, histidine,phenylalanine, arginine; the amino acid is preferably selected form abasic amino acid such as lysine, histidine or arginine; most preferablyarginine.

As used herein, the term “room temperature” refers to a temperature offrom 20° C. to 38° C.

As used herein, the term “RRT” refers to a retention time relative tothe retention time of albumin monomers retained in size exclusionchromatography-HPLC.

As used herein, the term “albumin monomer” refers to a single albuminmolecule without an intermolecular disulfide bond.

As used herein, the term “albumin dimer” refers to an albumin specieshaving a RRT of from about 0.86 to about 0.97 relative to the retentiontime of albumin monomers retained in size exclusion chromatography-HPLC.

As used herein or in the present invention, the term “albumin polymer”refers to the sum of all albumin species except for albumin monomers anddimers, i.e., albumin species having a RRT of from about 0.50 to about0.85 relative to the retention time of albumin monomers retained in sizeexclusion chromatography-HPLC.

As used herein, the term “taxanes” or “taxane compounds” include anyditerpene compounds that inhibit the depolymerization of tubulin,including those naturally occurring or synthetic, crystalline and/ornon-crystalline compounds, and the examples include, but are not limitedto, paclitaxel, docetaxel, and cabazitaxel as well as analogs,derivatives, and prodrug forms thereof. The analogs and/or derivativesmay include alcohols, ethers, esters, amines, salts, amides, halides,sulfides of paclitaxel, docetaxel and cabazitaxel or mixtures of two ormore thereof.

In view of the change in spatial structure of albumin nanoparticles dueto embedding a drug, the present inventors have surprisingly found thatthe addition of an amino acid having a relative molecular mass of145-175 (particularly the amino acid selected from one or more ofarginine, histidine and lysine, preferably arginine and/or histidine,more preferably arginine) or a salt thereof can effectively inhibit theincrease of albumin dimers and polymers during the storage of acorresponding albumin nanoparticle pharmaceutical composition, whereasaddition of other amino acid (e.g., glycine, aspartic acid etc.) haslittle effect on the inhibition of the increase of albumin dimers andpolymers during the storage of the albumin nanoparticle pharmaceuticalcomposition. Further, the addition of the amino acid or the salt thereofaccording to the present invention can effectively inhibit thedegradation of albumin monomers during the storage of the albuminpharmaceutical composition.

The inventors have also surprisingly found that, in an albuminnanoparticle pharmaceutical composition comprising a taxane compound,the addition of the amino acid may allow albumin monomers remain in anamount of no less than about 80% of the total amount of the albumins,and/or albumin dimers in an amount of no more than about 10%, and/oralbumin polymers in an amount of no more than about 6%. For example, theaddition of the amino acid, particularly arginine or a salt thereof, toa corresponding albumin nanoparticle pharmaceutical compositioncomprising paclitaxel may allow the content of the albumin monomersremain in above 80% of the total amount of the albumins, the albumindimers below 8%, and the polymers below 5%, after a storage of 24 monthsat room temperature (e.g., 25° C.). An addition of the amino acid havinga relative molecular mass of 145-175, particularly arginine or the saltthereof, to a corresponding albumin nanoparticle pharmaceuticalcomposition comprising docetaxel may allow the content of the albuminmonomers remain above 85% of the total amount of the albumins, thealbumin dimers below 5%, and albumin polymers below 5%, after a storageof 24 months at room temperature (e.g., 25° C.).

Based on the above discoveries, the present invention provides analbumin nanoparticle pharmaceutical composition, comprising a taxanecompound, an albumin and at least one amino acid having a relativemolecular mass of 145-175 (particularly the amino acid selected from oneor more of arginine, histidine and lysine, preferably arginine and/orhistidine, more preferably arginine) or a salt thereof as a stabilizer,wherein the composition comprises albumin monomers in an amount of noless than about 80% of the total amount of the albumins, and/or albumindimers in an amount of no more than about 10% of the total amount of thealbumins, and/or albumin polymers in an amount of no more than about 6%of the total amount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin monomers in an amount of noless than about 80%, about 85%, about 87%, about 90%, about 92%, about93% or higher, of the total amount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin dimers in an amount of nomore than about 10%, about 8%, about 6%, about 5% or less, of the totalamount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin polymers in an amount of nomore than about 6%, about 4%, about 2% or less, of the total amount ofthe albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin monomers in an amount of noless than about 80%, and/or albumin dimers in an amount of no more thanabout 9%, and/or albumin polymers in an amount of no more than about 6%,of the total amount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin monomers in an amount of noless than about 83%, and/or albumin dimers in an amount of no more thanabout 8%, and/or albumin polymers in an amount of no more than about 5%,of the total amount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin monomers in an amount of noless than about 85%, and/or albumin dimers in an amount of no more thanabout 5%, and/or albumin polymers in an amount of no more than about 5%,of the total amount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin monomers in an amount of noless than about 88%, and/or albumin dimers in an amount of no more thanabout 6%, and/or albumin polymers in an amount of no more than about 3%,of the total amount of the albumins.

In some embodiments, the albumin nanoparticle pharmaceutical compositionof the present invention comprises albumin monomers in an amount of noless than about 90%, and/or albumin dimers in an amount of no more thanabout 5%, and/or albumin polymers in an amount of no more than about 2%,of the total amount of the albumins.

The amino acid used in the present invention is selected from one ormore of arginine, histidine and lysine, preferably arginine and/orhistidine, more preferably arginine.

A salt of the amino acid according to the present invention may alsoachieve the same or similar effects as that achieved by the amino acid.The salt of the amino acid includes an inorganic acid salt and anorganic acid salt, for example, but is not limited to, hydrochloride,sulfate, nitrate etc., as well as carboxylate (e.g., formate, acetate),sulfonate, and the like. Examples of the salt of the amino acidaccording to the present invention includes, but is not limited to,arginine hydrochloride, arginine nitrate, histidine hydrochloride,histidine sulfate, histidine acetate, lysine sulfonate, lysine sulfate,and the like. In the present invention, the amount of a salt of an aminoacid is expressed by the amount of the amino acid after conversion.

The taxane compound used in the present invention is selected from oneor more of paclitaxel, docetaxel, cabazitaxel, and derivatives thereof.

The albumin used in the present invention is selected from one or moreof recombinant albumin, animal serum albumin, and human serum albumin,preferably human serum albumin.

In some embodiments, the human serum albumin used in the presentinvention may comprise a stabilizer such as sodium octanoate and/or asalt of acetyltryptophan etc. In other embodiments, the human serumalbumin used in the present invention may be substantially free ofstabilizers such as sodium octanoate and/or salts of acetyltryptophanetc.

The source of the albumin used in the present invention is not limited,and any modification or improvement of albumin is not required.

In some embodiments, in addition to the amino acid, other stabilizersuch as sodium octanoate and/or a salt of acetyl tryptophan may be addedto the albumin nanoparticle pharmaceutical composition of the presentinvention.

In some embodiments, the amino acid or the salt thereof is the solestabilizer of the albumin nanoparticle pharmaceutical composition of thepresent invention.

In some embodiments, the albumin nanoparticle pharmaceuticalcompositions of the present invention are substantially free oflyoprotectants.

In some embodiments, the weight ratio of the amino acid or the saltthereof to the albumin in the albumin nanoparticle pharmaceuticalcomposition of the present invention may be from 0.1:1 to 10:1, or from0.15:1 to 8:1, or from 0.2:1 to 5:1, or from 0.25:1 to 4:1, or from0.3:1 to 3:1, or from 0.4:1 to 2:1, or from 0.5:1 to 1:1.

In some embodiments, the weight ratio of the amino acid having arelative molecular mass of 145-175 or the salt thereof to the taxanecompound in the albumin nanoparticle pharmaceutical composition of thepresent invention may be no more than 80:1, or from 2:1 to 60:1, or from3:1 to 50:1, or form 3:1 to 20:1, or from 3:1 to 15:1, or the like. Insome embodiments, the weight ratio of the amino acid or the salt thereofto the taxane compound in the albumin nanoparticle pharmaceuticalcomposition of the present invention may range from 3:1 to 20:1, such asabout 4.5: 1, about 7.5:1, about 11.25:1, and the like.

In some embodiments of the albumin nanoparticle pharmaceuticalcomposition comprising docetaxel according to the present invention, theweight ratio of the amino acid (e.g., arginine) or the salt thereof todocetaxel may range from 2:1 to 50: 1, or from 3:1 to 30:1, or from 3:1to 20:1, or from 4:1 to 25:1, or from 4:1 to 20:1, or from 5:1 to 20:1,or from 8: 1 to 20:1, or from 10:1 to 15:1 etc., for example, about11.25:1.

In some embodiments of the albumin nanoparticle pharmaceuticalcomposition comprising paclitaxel according to the present invention,the weight ratio of the amino acid (e.g., arginine or histidine) or thesalt thereof to paclitaxel may be from 2:1 to 50:1, or from 2:1 to 30:1,or from 3:1 to 20:1, or from 3:1 to 15:1, or from 3:1 to 10:1 etc., forexample, about 4.5:1.

In some embodiments of the albumin nanoparticle pharmaceuticalcomposition comprising cabazitaxel according to the present invention,the weight ratio of the amino acid or the salt thereof (e.g., argininehydrochloride) to cabazitaxel may be from 1:1 to 50 :1, or from 2:1 to30:1, or from 3:1 to 25:1, or from 4:1 to 20:1, or from 5:1 to 15:1etc., for example, about 7.5:1.

In some embodiments, in the albumin nanoparticle pharmaceuticalcomposition according to the present invention, the weight ratio of theamino acid or the salt thereof to the albumin may be from 0.1:1 to 10:1,or from 0.15:1 to 8:1, or from 0.2:1 to 5:1, or from 0.25:1 to 4:1, orfrom 0.3:1 to 3:1, or from 0.4:1 to 2:1, or from 0.5:1 to 1:1, and theweight ratio of the amino acid or the salt thereof to the taxanecompound may be no more than 80:1, for example, from 2:1 to 60:1, orfrom 3:1 to 50:1, or from 3:1 to 20:1, or from 4:1 to 15:1 etc., forexample, about 4.5:1, or about 7.5:1, or about 11.25:1, and the like.

In some embodiments, the albumin nanoparticle pharmaceutical compositionaccording to the present invention comprises paclitaxel, a human serumalbumin, and arginine and/or histidine, wherein the weight ratio of thearginine and/or histidine to the human serum albumin is from 0.2:1 to5:1, preferably from 0.3:1 to 3:1, and the weight ratio of arginineand/or histidine to paclitaxel is from about 3:1 to 20:1, preferablyfrom 3:1 to 10:1.

In some embodiments, the albumin nanoparticle pharmaceutical compositionaccording to the present invention comprises docetaxel, a human serumalbumin, and arginine, wherein the weight ratio of arginine to the humanserum albumin is from 0.2:1 to 5:1, preferably from 0.3:1 to 3:1, andthe weight ratio of arginine to docetaxel is from about 3:1 to 20:1,preferably from 5:1 to 20:1.

In some embodiments, the albumin nanoparticle pharmaceutical compositionaccording to the present invention comprises cabazitaxel, a human serumalbumin, and arginine hydrochloride, wherein the weight ratio of thearginine hydrochloride (expressed by the weight of arginine) to thehuman serum albumin is from 0.2:1 to 5:1, preferably from 0.3:1 to 3:1,and the weight ratio of arginine hydrochloride (expressed by weight ofarginine) to cabazitaxel is about from 3:1 to 20:1, preferably from 5:1to 15:1.

The nanoparticle of the albumin nanoparticle pharmaceutical compositionof the present invention has an average particle diameter of not morethan 200 nm. In some embodiments, the nanoparticle of the albuminnanoparticle pharmaceutical composition of the present invention has anaverage particle size of not more than 160 nm. In some embodiments, thenanoparticle of the albumin nanoparticle pharmaceutical composition ofthe present invention has an average particle size of no more than 130nm. In some embodiments, the nanoparticle of the albumin nanoparticlepharmaceutical composition of the present invention has an averageparticle size of not more than 110 nm. In some embodiments, thenanoparticle of the albumin nanoparticle pharmaceutical composition ofthe present invention has an average particle size of, for example,about 95 nm, about 100 nm, about 110 nm, about 120 nm.

The albumin nanoparticle composition of the present invention comprisesone or more of the above characteristics.

It is an object of the present invention to provide a method forstabilizing albumin nanoparticle, comprising reducing albumin dimersand/or polymers or inhibiting their formation/increase by using anappropriate amount of at least one amino acid having a relativemolecular mass of 145-175 (particularly the amino acid selected from oneor more of arginine, histidine and lysine, preferably arginine and/orhistidine, more preferably arginine) or a salt thereof. The method ofthe present invention, comprising using the amino acid or the saltthereof, thereby reducing the formation of albumin dimers and/orpolymers, improving the chemical stability of albumin nanoparticle,consequently solving the problem of the physical and chemicalinstability of albumin nanoparticle fundamentally, reducing the typesand amount of impurities, reducing toxicity in vitro and in vivo,improving the applicability in patients.

Emulsification through high pressure homogenization or the like isapplied in the method of the present invention, which comprises addingan appropriate amount of the amino acid of the present invention or thesalt thereof during the preparation of albumin nanoparticle comprising adrug and a carrier of albumin under high shear condition.

The present invention particularly provides a method for preparing analbumin nanoparticle pharmaceutical composition, comprising dispersing ataxane compound in an organic solvent to obtain an organic phase,dispersing an albumin in an aqueous solvent to obtain an aqueous phase,dispersive-mixing the organic phase and the aqueous phase under highshear force to obtain a nanoparticle solution, adding a solution of theamino acid of the present invention in the above procedure to obtain aresulting solution, and subjecting the resulting solution tosterilization by filtration and optional lyophilization to obtain thealbumin nanoparticle pharmaceutical composition or lyophilizedformulation thereof.

The present invention particularly provides a preparation method,comprising the following steps: dissolving a taxane compound in asuitable organic solvent to obtain a solution of a taxane compound;dissolving or diluting an albumin into an aqueous solvent to obtain anaqueous solution of albumin; preparing an aqueous solution of an aminoacid having a relative molecular mass of 145-175 or a salt thereof byusing water (preferably, water for injection), wherein the amino acidparticularly refers to one or more amino acid selected from arginine,histidine, and lysine, preferably arginine and/or histidine, morepreferably arginine; and subjecting the solution of the taxane compound,the aqueous solution of the albumin and the aqueous solution of theamino acid having a relative molecular mass of 145-175 or the saltthereof to dispersive-mixing under high shear force, thenultrafiltration, sterilization by filtration, and optionallyophilization.

In some embodiments, a method for preparing the albumin nanoparticlepharmaceutical composition according to the present invention isprovided, wherein the method comprising the steps of:

the taxane compound is dissolved in a suitable organic solvent (e.g.,ethanol) to obtain a solution of the taxane compound; the albumin isdissolved or diluted into an aqueous solvent (e.g., water), and thenincubated, for example, incubated in a water bath at a temperature of55-75° C. for 3-15 minutes, to obtain an aqueous solution of the albumin(e.g., aqueous solution); an aqueous solution of the amino acid or thesalt thereof (e.g., an aqueous solution having a pH of 6.5-7.5) of thepresent invention is prepared with an acid and/or base (e.g.,hydrochloric acid and/or sodium hydroxide) as a pH regulator; thesolution of taxane compound, the aqueous solution of the albumin and theaqueous solution of the amino acid or the salt thereof are subjected todispersive-mixing under high shear force (for example, at 1000-8000 rpm)and then ultrafiltration to obtain a concentrated solution of thealbumin nanoparticle; and the concentrated solution is sterilized byfiltration and optionally lyophilized.

In the above process, the weight ratio of the amino acid or the saltthereof to albumin is from 0.1:1 to 10:1.

In the above preparation method, the water used is preferably water forinjection.

A person skilled in the art can carry out the above steps in a differentorder to prepare the albumin nanoparticle pharmaceutical composition ofthe present invention. For example, the aqueous solution of the aminoacid or the salt thereof, the solution of the taxane compound, and theaqueous solution of the albumin can be prepared separately, and theaqueous solution of the amino acid or the salt thereof, the aqueoussolution of the albumin, and the solution of the taxane compound aremixed under high shear force, then ultrafiltered, sterilized byfiltration, and consequently lyophilized.

When the method of the present application is applied, it is notnecessary to add a lyoprotectant, such as one or more lyoprotectantsselected from trehalose, sucrose, maltose, lactose and glycine, prior tothe lyophilization.

The amino acid or the salt thereof is added to the albumin nanoparticlepharmaceutical composition by the preparation method of the presentinvention to obtain the albumin nanoparticle pharmaceutical compositionhaving satisfactory effect in inhibiting the increase of albumin dimersand polymers, as well as in inhibiting the degradation of albuminmonomers.

In another aspect, the present invention provides a pharmaceuticalformulation comprising the albumin nanoparticle pharmaceuticalcomposition of the present invention.

In some embodiments, the pharmaceutical formulation of the presentinvention may further comprise a second active ingredient/secondtherapeutic agent. The second active ingredient/second therapeutic agentused in the present invention may include, but is not limited to, ananthracycline drug (e.g., epirubicin), a nucleoside compound (e.g.,gemcitabine), and the like.

In some embodiments, the pharmaceutical formulation of the presentinvention further comprises one or more pharmaceutically acceptablecarriers and/or adjuvants.

In some embodiments, the pharmaceutically acceptable carrier and/oradjuvant used in the present invention include sterile water, saline,dextrose, oil (e.g., corn oil, peanut oil, sesame oil, and the like),acid, lower alkanols (glycol; polyalkylene glycol). In otherembodiments, the pharmaceutically acceptable carrier and/or adjuvantused in the present invention may also include preservatives, wettingagents, emulsifying agents, penetration enhancers, and the like.

For example, in some embodiments, the composition/pharmaceuticalformulation of the present invention may further comprise anantimicrobial agent (e.g., a chelating agent, which includes, but is notlimited to, EDTA, edetate(ester), citrate(ester), pentetate(ester),tromethamine, sorbate(ester), ascorbate(ester), a derivative thereof, ora mixture thereof; e.g., a non-chelating agent, which includes, but isnot limited to, any one or more of sulfite, benzoic acid, benzylalcohol, chlorobutanol and paraben), a sugar (e.g., sucrose, and thelike), other reconstruction enhancer (such as those described in U.S.Patent Application having a publication number of 2005/0152979, and allcontents of which are incorporated herein by reference), a negativelycharged component (e.g., bile salts, bile acids, glycocholic acid,cholic acid, chenodeoxycholic acid, taurocholic acid,glycochenodeoxycholic acid, taurochenodeoxycholic acid, lithocholicacid, ursodeoxycholic acid, dehydrocholic acid, phospholipids).

In some embodiments, the pharmaceutical formulation of the presentinvention is a lyophilized powder injection. The nanoparticlepharmaceutical composition of the present invention can be prepared intolyophilized powder injection by a preparation method well known in theart.

The albumin nanoparticle pharmaceutical composition of the presentinvention can be used for treating a cancer, including, but not limitedto, one or more selected from the group consisting of prostate cancer,colon cancer, breast cancer (e.g., metastatic breast cancer), head andneck cancer, pancreatic cancer (e.g., metastatic pancreatic cancer orlocally advanced non-resectable pancreatic cancer), lung cancer (e.g.,non-small cell lung cancer), ovarian cancer, multiple myeloma, renalcell carcinoma, melanoma (e.g., metastatic melanoma), liver cancer,gastric cancer, and kidney cancer.

The administration dose of the albumin nanoparticle pharmaceuticalcomposition of the present invention may vary depending on a particulartaxane compound, administration method, and particular type of thecancer to be treated. This dose is sufficient to produce the desiredbenefits. The compositions can be formulated for single- ormultiple-dose administration.

The albumin nanoparticle pharmaceutical composition or pharmaceuticalformulation of the present invention can be administered by any routeknown to a person skilled in the art, including intramuscular,intravenous, intradermal, intralesional, intraperitoneal injection orany other suitable route. The administration can be topical, topical orsystemic administration, depending on a site to be treated. The mostappropriate route in any given situation depends on a variety offactors, such as the nature, progression and severity of a disease, andthe like. In some embodiments, the pharmaceutical composition orpharmaceutical formulation of the present invention is administered byintravenous injection.

In some embodiments, the nanoparticle pharmaceutical composition orpharmaceutical formulation of the present invention may also beadministered with a second active ingredient/second therapeutic agent.In some embodiments, the nanoparticle pharmaceutical composition orpharmaceutical formulation of the present invention can be administeredsimultaneously, sequentially or in parallel with the second activeingredient/second therapeutic agent. When administered separately, thenanoparticle pharmaceutical composition or pharmaceutical formulation ofthe present invention and the second active compound or secondtherapeutic agent can be administered at different frequency or intervalof administration.

The second active ingredient/second therapeutic agent used in thepresent invention may include, but is not limited to, an anthracyclinedrug (e.g., epirubicin), a nucleoside compound (e.g., gemcitabine), andthe like.

In another aspect, the present invention provides use of the albuminnanoparticle pharmaceutical composition or pharmaceutical formulation ofthe present invention in the manufacture of a medicament for thetreatment of a disease associated with cell proliferation or cellhyperproliferation (e.g., cancer).

In some embodiments, the cancer includes, but is not limited to, one ormore selected from the group consisting of prostate cancer, coloncancer, breast cancer (e.g., metastatic breast cancer), head and neckcancer, pancreatic cancer (e.g., metastatic pancreatic cancer or locallyadvanced non-resectable pancreatic cancer), lung cancer (e.g., non-smallcell lung cancer), ovarian cancer, multiple myeloma, renal cellcarcinoma, melanoma (e.g., metastatic melanoma), liver cancer, gastriccancer, and kidney cancer.

In some embodiments, the medicament is a lyophilized powder injection.The lyophilized powder injection can be re-formulated as a solution, anemulsion or a suspension for administration, or can be formulated into acolloid.

In another aspect, the invention provides use of an amino acid having arelative molecular mass of 145-175 (particularly the amino acid selectedfrom one or more of arginine, histidine, and lysine, preferably arginineand/or histidine, more preferably arginine) or a salt thereof forinhibiting the content of albumin dimers and/or polymers in an albuminnanoparticle pharmaceutical composition (particularly the albuminnanoparticle pharmaceutical composition of the present invention) duringstorage. In some embodiments, the weight ratio of the amino acid havinga relative molecular mass of 145-175 or the salt thereof to albumin inthe pharmaceutical composition is from 0.1:1 to 10:1, preferably from0.2:1 to 5:1.

In some embodiments, the storage period may be 24 months for which thestorage is performed at room temperature (e.g., 25° C.).

As compared with a composition without the addition of the amino acid orthe salt thereof of the present invention, the taxane-albuminnanoparticle composition added with the amino acid or a salt thereof hassignificantly improved the albumin characteristics and a reducedpolymerization rate of the albumin. This reduced ratio can be evaluated,for example, under conditions of long-term storage at room temperature(e.g., 25° C.) or under accelerated conditions at an elevatedtemperature (e.g., 60° C.).

In some embodiments, the addition of the amino acid, e.g., arginine, ofthe present invention or the salt thereof to the corresponding albuminnanoparticle pharmaceutical composition (e.g., an albumin nanoparticlepharmaceutical composition comprising paclitaxel) may allow albumindimers and polymers to be controlled at no more than about 8% and about5% of the total amount of the albumins, respectively, and albuminmonomers to be maintained at no less than 83% of the total amount of thealbumins, under conditions of 24 months storage at room temperature(e.g., 25° C.).

In some embodiments, the addition of the amino acid, e.g., arginine, ofthe present invention or the salt thereof to the corresponding albuminnanoparticle pharmaceutical composition (e.g., an albumin nanoparticlepharmaceutical composition comprising docetaxel) may allow both ofalbumin dimers and polymers to be controlled at no more than about 5% ofthe total amount of the albumins, respectively, and albumin monomers tobe maintained at no less than 85% of the total amount of the albumins,under conditions of 24 months storage at room temperature (e.g., 25°C.).

In some embodiments, the addition of the amino acid, e.g., histidine, ofthe present invention or the salt thereof to the corresponding albuminnanoparticle pharmaceutical composition (e.g., an albumin nanoparticlepharmaceutical composition comprising paclitaxel) may allow albumindimers and polymers to be controlled at no more than about 9% and about6% of the total amount of the albumins, respectively, and albuminmonomers to be maintained at no less than 80% of the total amount of thealbumins, under conditions of 10 days storage at an elevated temperature(e.g., 60° C.).

In some embodiments, the addition of the amino acid, e.g., arginine, ofthe present invention or the salt thereof to the corresponding albuminnanoparticle pharmaceutical composition (e.g., an albumin nanoparticlepharmaceutical composition comprising paclitaxel) may allow albumindimers and polymers to be controlled at no more than about 6% and about3% of the total amount of the albumins, respectively, and albuminmonomers to be maintained at no less than 88% of the total amount of thealbumins, under conditions of 10 days storage at an elevated temperature(e.g., 60° C.).

In some embodiments, the addition of the amino acid, e.g., arginine, ofthe present invention or the salt thereof to the corresponding albuminnanoparticle pharmaceutical composition (e.g., an albumin nanoparticlepharmaceutical composition comprising cabazitaxel) may allow albumindimers and polymers to be controlled at no more than about 5% and about2% of the total amount of the albumins, respectively, and albuminmonomers to be maintained at no less than 90% of the total amount of thealbumins, under conditions of 10 days storage at an elevated temperature(e.g., 60° C.).

In another aspect, the present invention provides use of an amino acidhaving a relative molecular mass of 145-175 (particularly the amino acidselected form one or more of arginine, histidine, and lysine, preferablyarginine and/or histidine, more preferably arginine) or a salt thereofin the manufacture an albumin nanoparticle pharmaceutical compositionfor reducing adverse reactions in a subject, wherein the albuminnanoparticle pharmaceutical composition comprises a taxane compound, andin the pharmaceutical composition, albumin monomers is in an amount ofno less than about 80% of the total amount of the albumins, and/oralbumin dimers is in an amount of no more than about 10% of the totalamount of the albumins, and/or albumin polymers is in an amount of nomore than about 6% of the total amount of the albumins.

In some embodiments, the amino acid of the present invention or the saltthereof is used as a stabilizer, further as the sole stabilizer, in thealbumin nanoparticle pharmaceutical composition.

The beneficial effects of the present invention are as follows:

1. By adding the amino acid having a relative molecular mass of 145-175(particularly the amino acid selected from one or more of arginine,histidine and lysine, preferably arginine and/or histidine, morepreferably arginine) or the salt thereof to the albumin pharmaceuticalcomposition, the degradation of albumin monomers and the increase ofalbumin dimers and polymers in the composition during storage iseffectively inhibited, and the adverse reactions in clinicalapplications thereof, such as rash, urticaria, anaphylactic reaction andpossible immune response, are effectively reduced, further ensuring thesafety in clinical medication.

2. No additional stabilizer is required to be added, and there is noneed to limit the source or make any modification or improvement to thealbumin in the pharmaceutical composition; correspondingly, the rawmaterial sources are wide and the preparation method is simple.

The present invention is further illustrated in the following examples.It is necessary to be noted that the following examples should not to beunderstood as any limitation of the protection scope of the presentinvention, and some non-essential improvements and adjustments to thepresent invention in light of the contents of the present inventiondescribed above made by a person skilled in the art still fall withinthe protection scope of the present invention.

The Abraxane used in the examples was a paclitaxel albumin nanoparticleformulation purchased from Celgene. The docetaxel, paclitaxel andcabazitaxel are active pharmaceutical ingredient, which are commerciallyavailable or can be prepared by methods well known to a person skilledin the art.

EXAMPLE 1 Preparation of a Docetaxel-Albumin Nanoparticle Composition byUsing Arginine

9 g Arginine was weighed, then an appropriate amount of water forinjection was added, the pH was adjusted to 6.8 with hydrochloric acidand/or NaOH, and then volumed to 30 ml to obtain a solution of arginine.Then, 0.8 g of docetaxel was added to a 100 ml beaker, followed by anaddition of 20 ml of ethanol, and ultrasonically dissolved to obtain anorganic phase comprising docetaxel. Human serum albumin was dissolved inwater for injection to prepare a 4 mg/ml solution, and incubated in awater bath at 63° C. for 10 minutes to obtain an aqueous phasecomprising human serum albumin. The organic phase comprising docetaxelwas uniformly dispersed in the aqueous phase comprising human serumalbumin under high-speed shear at 6,000 revolutions per minute (rpm),and the resulting dispersion was transferred to a device forconcentration by ultrafiltration and ultrafiltered to obtain a docetaxelnanoparticle solution with a docetaxel concentration of 5 mg/ml. Thesolution of arginine was added to the docetaxel nanoparticle solutionand stirred to obtain a docetaxel nanoparticle solution comprisingarginine, and the docetaxel nanoparticle solution comprising argininehad an average particle size of 153 nm measured by Malvern Nano-ZS90nanometer particle size analyzer. The prepared docetaxel nanoparticlesolution comprising arginine was subjected to filtration sterilizationwith a 0.22 μm sterile filter, and lyophilized for 48 hours to removeethanol and water for injection to obtain a powdery docetaxel-albuminnanoparticle composition.

EXAMPLE 2 Preparation of a Paclitaxel-Albumin Nanoparticle Compositionby Using Arginine

4.5 g Arginine was weighed, then an appropriate amount of water forinjection was added, the pH was adjusted to 7.0 with hydrochloric acidand/or NaOH, and then volumed to 15 ml to obtain a solution of arginine.Then, 1.0 g of paclitaxel was added to a 100 ml beaker, followed by anaddition of 25 ml of ethanol, and ultrasonically dissolved to obtain anorganic phase comprising paclitaxel. 9 g of human serum albumin wasdissolved in water for injection to prepare a 6 mg/ml solution, andincubated in a water bath at 65° C. for 10 minutes to obtain an aqueousphase comprising human serum albumin. The organic phase comprisingpaclitaxel was uniformly dispersed in the aqueous phase comprising humanserum albumin under high-speed shear at 3,000 revolutions per minute(rpm), and the dispersion was transferred to a device for concentrationby ultrafiltration and ultrafiltered to obtain a paclitaxel nanoparticlesolution with a paclitaxel concentration of 6 mg/ml. The solution ofarginine was added to the paclitaxel nanoparticle solution and stirredto obtain a paclitaxel nanoparticle solution comprising arginine, andthe paclitaxel nanoparticle solution comprising arginine had an averageparticle size of 123 nm measured by Malvern Nano-ZS90 nanometer particlesize analyzer. The prepared paclitaxel nanoparticle solution comprisingarginine was subjected to filtration sterilization with a 0.22 pmsterile filter, and lyophilized for 48 hours to remove ethanol and waterfor injection to obtain a powdery paclitaxel-albumin nanoparticlecomposition.

EXAMPLE 3 Preparation of a Paclitaxel-Albumin Nanoparticle Compositionby Using Histidine

A paclitaxel-albumin nanoparticle composition of Example 3 was preparedby a method similar to that of Example 2 except that histidine was usedas a stabilizer instead of arginine.

EXAMPLE 4 Preparation of a Cabazitaxel-Albumin Nanoparticle Compositionby Using Arginine Hydrochloride

4.5 g Arginine hydrochloride by weight of arginine was weighed, then anappropriate amount of water for injection was added, the pH was adjustedto 6.8 with hydrochloric acid and/or NaOH, and then volumed to 10 ml toobtain a solution of arginine hydrochloride. Then, 0.6 g of cabazitaxelwas added to a 100 ml beaker, followed by an addition of 20 ml ofethanol, and ultrasonically dissolved to obtain an organic phasecomprising cabazitaxel. Human serum albumin was dissolved in water forinjection to prepare a 4 mg/ml solution, and incubated in a water bathat 63° C. for 5 minutes to obtain an aqueous phase comprising humanserum albumin. The solution of arginine hydrochloride was added to theaqueous phase comprising human serum albumin to obtain an aqueous phasecomprising human serum albumin added with arginine hydrochloride. Theorganic phase comprising cabazitaxel was uniformly dispersed into theaqueous phase comprising human serum albumin added with argininehydrochloride under high-speed shear at 3,000 revolutions per minute(rpm), and the dispersion was transferred to a device for concentrationby ultrafiltration and ultrafiltered to be a cabazitaxel concentrationof 5 mg/ml to obtain a cabazitaxel nanoparticle solution comprisingarginine hydrochloride. The cabazitaxel nanoparticle solution comprisingarginine hydrochloride had an average particle size of 106 nm measuredby Malvern Nano-ZS90 nanometer particle size analyzer. The preparedcabazitaxel nanoparticle solution comprising arginine hydrochloride wassubjected to filtration sterilization with a 0.22 μm sterile filter, andlyophilized for 48 hours to remove ethanol and water for injection toobtain a powdery cabazitaxel -albumin nanoparticle composition.

COMPARATIVE EXAMPLE 1 Preparation of a Docetaxel-Albumin NanoparticleComposition without Arginine

A docetaxel albumin nanoparticle composition was prepared by a methodsimilar to that of Example 1 except that the solution of arginine wasnot added.

COMPARATIVE EXAMPLE 2 Preparation of a Paclitaxel-Albumin NanoparticleComposition without Arginine

A paclitaxel-albumin nanoparticle composition was prepared by a methodsimilar to that of Example 2 except that the solution of argininesolution was not added.

COMPARATIVE EXAMPLE 3 Preparation of a Paclitaxel-Albumin Nanoparticlecomposition by Using Glycine

A paclitaxel-albumin nanoparticle composition was prepared by a methodsimilar to that of Example 2 except that glycine was used instead ofarginine.

COMPARATIVE EXAMPLE 4 Preparation of a Paclitaxel-Albumin NanoparticleComposition by Using Aspartic Acid

A paclitaxel-albumin nanoparticle composition was prepared by a methodsimilar to that of Example 2 except that aspartic acid was used insteadof arginine.

COMPARATIVE EXAMPLE 5 Preparation of a Cabazitaxel-Albumin NanoparticleComposition without Arginine Hydrochloride

A cabazitaxel-albumin nanoparticle composition was prepared by a methodsimilar to that of Example 4 except that the solution of argininehydrochloride was not added.

COMPARATIVE EXAMPLE 6 Preparation of a Cabazitaxel-Albumin NanoparticleComposition by Using Sodium Octanoate as a Stabilizer

A cabazitaxel-albumin nanoparticle composition was prepared by a methodsimilar to that of Example 4 except that sodium octanoate was usedinstead of arginine.

COMPARATIVE EXAMPLE 7 Preparation of a Cabazitaxel-Albumin NanoparticleComposition by Using Sodium Octanoate and Acetyl Tryptophan asStabilizer

A cabazitaxel-albumin nanoparticle composition was prepared by a methodsimilar to that of Example 4 except that sodium octanoate and acetyltryptophan (1:1) were used as a stabilizer instead of arginine.

TEST EXAMPLE 1

The increase tendency of albumin dimers and polymers as well as thedegradation tendency of albumin monomers in the docetaxel-albuminnanoparticle composition prepared according to the methods described inExample 1 and Comparative Example 1 was observed in the study oflong-term stability under conditions of 24 months storage at 25° C. Thecontent of albumin monomers, dimers and polymers in the nanoparticlecomposition were analyzed by size exclusion chromatography.

Conditions of size exclusion HPLC were as follows:

Chromatographic conditions and system suitability test: a size exclusionchromatography containing hydrophilic silica gel (the chromatographycolumn was: TOSOH TSK gel G3000SWxl, 300×7.8 mm 5 μm; and the guardcolumn was: TOSOH TSK gel G3000 SWxl, 40×6.0 mm 7 μm) was employed,wherein a 0.2 mol/L and pH 7.0 phosphate buffer solution containing 1%isopropanol (the solution was obtained by mixing 200 ml of 0.5 mol/Lsodium dihydrogen phosphate, 420 ml of 0.5 mol/L disodium hydrogenphosphate, 15.5 ml of isopropanol and 914.5 ml of water) was used asmobile phase; the test wavelength was 280 nm; the flow rate was 0.6ml/min; and the column temperature was 30° C.

Determination method: an appropriate amount of a reference substance ofhuman blood albumin was taken and diluted with water to prepare asolution containing about 6.4 mg, 3.2 mg, or 1.6 mg of albumin per 1 mlas a reference solution. 20 μl of each of the reference solutions wasmeasured off and injected into the HPLC liquid chromatography, and thecorresponding chromatograms were recorded. The linear regressionequation was calculated from the concentration and peak area of thereference solution.

80 mg of each of the compositions obtained in Example 1 and ComparativeExample 1 was taken as test samples and placed in a 25 ml volumetricflask, dissolved in water and diluted to the mark, and shaken well toprepare a solution with the albumin concentration of about 3 mg/ml. 20μl of each of the test sample solutions was measured off, and injectedinto the HPLC liquid chromatograph, and the corresponding chromatogramswere recorded. The content of monomers, dimers and polymers of humanserum albumin in the test samples were calculated according to theregression equation.

Calculation Manner:

The content of albumin dimers refers to a percentage of the peak areawith a retention time RRT of from about 0.86 to about 0.97 with respectto the entire peak area in the HPLC chromatogram.

The content of albumin polymers refers to a value obtained by dividing apercentage of the peak area with a retention time RRT of from about 0.50to about 0.85 with respect to the entire peak area in the size exclusionHPLC chromatogram by two.

The content of albumin monomer was calculated using the followingformula: albumin monomers %=1-2×albumin polymers %—albumin dimers %.

Test results were shown in Table 1.

TABLE 1 contents of albumin monomers, dimers and polymers in thecomposition comprising arginine of Example 1 and in the compositionwithout arginine of Comparative Example 1 in the study on long-termstability at 25° C. Composition Example 1 Comparative Example 1 25° C.monomers dimers polymers monomers dimers polymers 0^(th) day 91.2% 2.0%3.4% 90.0%  2.8% 3.6% 24 86.4% 4.6% 4.5% 71.2% 17.2% 5.8% months

The results in Table 1 showed that: in the study on the long-termstability at 25° C., in the docetaxel-albumin nanoparticle compositionof Example 1, with the addition of arginine, the content of albuminmonomers could be maintained at more than 85% for a long period of time,the content of albumin dimers could be maintained at no more than 5% fora long period of time, and the content of albumin polymers could bemaintained at no more than 5% for a long period of time, while in thecomposition without arginine of Comparative Example 1, the content ofalbumin monomers was only maintained at about 70%, and the content ofalbumin dimers and polymers were significantly more than 5%.

Apparently, with the addition of the amino acid of the presentinvention, particularly arginine, the albumin dimers and polymers in thedocetaxel-albumin nanoparticle composition could be significantlyreduced, and the albumin monomers were effectively stabilized, thedegradation of albumin monomers was substantially avoided, therebyensuring the safety in clinical application.

TEST EXAMPLE 2

The content of albumin monomers, dimers and polymers in thepaclitaxel-albumin nanoparticle compositions prepared according toExample 2 and Comparative Example 2 and in the Abraxane in the long-termstability study thereof under conditions of 24 months storage at 25° C.was determined by the method in Text Example 1. The composition ofExample 2 was the paclitaxel-albumin nanoparticle composition comprisingarginine, the composition of Comparative Example 2 was thepaclitaxel-albumin nanoparticle composition without arginine, and thecommercially available Abraxane sample was prepared by the method sameas the method for the preparation of the compositions of Example 2 andComparative Example 2. Test results were shown in Table 2.

TABLE 2 Content of albumin monomers, dimers and polymers in thecomposition of Example 2 and Comparative Example 2 in the study onlong-term stability at 25° C. Composition Example 2 Comparative Example2 conditions monomers dimers polymers monomers dimers polymers 25° C.0^(th) day 92.0% 4.3% 1.8% 91.0%  4.6% 2.2%  6 months 88.7% 4.5% 3.4%78.0% 16.2% 5.8% 12 months 86.4% 5.8% 3.9% 64.0% 17.8% 9.1% 24 months84.0% 7.4% 4.3% 58.2% 20.9% 11.5% 

In addition, after a storage under the above conditions for 24 months,the contents of albumin monomers, dimers, and polymers in thecommercially available Abraxane were 72.6%, 15.2%, and 6.1%,respectively.

The results in Table 2 showed that: in the study on the long-termstability at 25° C., in the paclitaxel-albumin nanoparticle composition,with the addition of arginine, the content of albumin monomers could bemaintained at more than 80% for a long period of time, the content ofalbumin dimers could be maintained at no more than 8% for a long periodof time, and the content of albumin polymers could be maintained at nomore than 5% for a long period of time, while in Abraxane and thecomposition without arginine, i.e., the composition of ComparativeExample 2, the contents of albumin dimers and polymers weresignificantly more than 5%, the content of albumin monomers in theAbraxane was only maintained at about 70% of the total amount of thealbumins, and the content of albumin monomers in the composition ofComparative Example 2 without arginine was less than 60% of the totalamount of the albumins.

Apparently, with the addition of the amino acid of the presentinvention, particularly arginine, the contents of albumin dimers andpolymers in the paclitaxel-albumin nanoparticle composition could besignificantly reduced, even the increase of the albumin dimers andpolymers during storage could be inhibited, and the albumin monomerswere effectively stabilized, the degradation of albumin monomers wassubstantially avoided, thereby ensuring the safety in clinicalapplication..

TEST EXAMPLE 3

The increase tendency of albumin dimers and polymers in thepaclitaxel-albumin nanoparticle compositions prepared according to themethods described in Examples 2 and 3 and Comparative Examples 2 to 4under accelerated conditions at 60° C. was observed. The contents ofalbumin monomers, dimers and polymers in the nanoparticle compositionswere determined by size exclusion chromatography. The conditions,determination method and calculation manner of the size exclusion HPLCwere same as those in Test Example 1. The composition in Example 2 was apaclitaxel-albumin nanoparticle composition comprising arginine, thecomposition in Example 3 was a paclitaxel-albumin nanoparticlecomposition comprising histidine, the composition in Comparative Example2 was a paclitaxel-albumin nanoparticle composition without arginine,the composition in Comparative Example 3 was a paclitaxel-albuminnanoparticle composition comprising glycine, and the composition inComparative Example 4 is a paclitaxel-albumin nanoparticle compositioncomprising aspartic acid. Test results were shown in Tables 3-1 and 3-2.

TABLE 3-1 Contents of albumin monomers, dimers and polymers in thecompositions of Examples 2-3 and the compositions of ComparativeExamples 2-4 under accelerated conditions at 60° C. conditions 60° C.compo- 0^(th) day 10^(th) day sition monomers dimers polymers monomersdimers polymers Example 92.0% 4.3% 1.8% 90.2%  5.4%  2.2% 2 Example91.8% 4.3% 1.9% 80.1%  8.3%  5.8% 3 Compar- 92.0% 4.1% 1.9% 70.6% 15.9% 6.8% ative Example 2 Compar- 92.2% 4.5% 1.7% 60.8% 18.2% 10.5% ativeExample 3 Compar- 91.9% 4.0% 2.5% 65.3% 17.6%  8.6% ative Example 4

TABLE 3-2 Change in contents of albumin monomers, dimers and polymers inthe compositions of Examples 2-3 and the compositions of ComparativeExamples 2-4 under accelerated conditions at 60° C. Reduced contentIncreased content Increased content of albumin of albumin of albuminExample monomers dimers polymers Example 2  1.8%  1.1% 0.4% Example 311.7%  4.0% 3.9% Comparative 21.4% 11.8% 4.9% Example 2 Comparative31.4% 13.7% 8.8% Example 3 Comparative 26.6% 13.6% 6.1% Example 4Note: the reduced content of albumin monomers =the content thereof onthe 0^(th) day−the content thereof on the 10^(th) day

the increased content of albumin dimers or albumin polymers=the contentsthereof on the 10^(th) day−the contents thereof on the 0^(th) day

The results showed that: under the accelerated conditions at 60° C.,with the addition of the amino acid having the relative molecular massdefined in the present invention, such as arginine (Example 2) andhistidine (Example 3), the contents of albumin polymers and albumindimers in the albumin nanoparticle compositions were maintained at lessthan 6% and 9% of the total amount of the albumins, respectively, thecontents were significantly lower than that of albumin polymers andalbumin dimers in the composition without arginine (Comparative Example2), and the compositions comprising an amino acid having a relativemolecular mass not fall in 145-175 such as glycine (Comparative Example3) and aspartic acid (Comparative Example 4). Further, the increasedamounts of albumin polymers and dimers in the compositions of Examples 2and 3 of the present invention were also significantly lower than thosein the compositions of Comparative Examples 2-4, meanwhile, the changedamounts of albumin monomers in the compositions of Examples 2 and 3 ofthe present invention were also significantly lower than that in thecompositions of Comparative Examples 2-4. These results demonstratedthat, under same conditions, the addition of the amino acid of thepresent invention could significantly reduce the increase of albumindimers and polymers and effectively stabilize albumin monomers in thealbumin nanoparticle composition, keep albumin monomers from beingdegraded substantially, thereby ensuring the safety in clinicalapplication.

TEST EXAMPLE 4

The contents of albumin monomers, dimers and polymers in thecabazitaxel-albumin nanoparticle composition comprising argininehydrochloride obtained in Example 4 and in the cabazitaxel-albuminnanoparticle compositions of Comparative Examples 5-7 were determinedunder the accelerated conditions at 60° C. by using the same method asin Test Example 3. Test results were shown in Table 4.

TABLE 4 Contents of albumin monomers, dimers and polymers in thecomposition of Example 4 and in the compositions of Comparative Examples5-7 at 60° C. Conditions 60° C. compo- 0^(th) day 10^(th) day sitionmonomers dimers polymers monomers dimers polymers Example 93.4% 4.2%1.2% 91.6%  4.8% 1.8% 4 Example 93.1% 4.1% 1.4% 71.2% 16.4% 6.2% 5Example 92.8% 4.1% 1.6% 76.6% 13.6% 4.9% 6 Example 92.5% 4.3% 1.6% 73.8%15.2% 5.5% 7

Apparently, with the addition of arginine hydrochloride, in thecabazitaxel-albumin nanoparticle composition of Example 4 of the presentapplication, the contents of albumin dimers and polymers weresignificantly lower than those in the corresponding composition withoutthe addition of arginine hydrochloride (Comparative Example 5), thecomposition with the addition of sodium octanoate (Comparative Example6) and the composition with the addition of sodium octanoate and acetyltryptophan (Comparative Example 7), the increased amounts of albumindimers and polymers were also significantly lower than those in thecorresponding composition without the salt of amino acid of the presentinvention, the composition with the addition of sodium octanoate and thecomposition with the addition of sodium octanoate and acetyl tryptophan,and the degradation rate of albumin monomers was significantly lowerthan that in the corresponding composition without the salt of aminoacid having a relative molecular mass of 145-175 of the presentinvention, the composition with the addition of sodium octanoate and thecomposition with the addition of sodium octanoate and acetyl tryptophan.These results demonstrated that, under same conditions, the addition ofthe salt of amino acid of the present invention could effectivelyinhibit the increase of albumin dimers and polymers.

TEST EXAMPLE 5

The contents of albumin monomers, dimers and polymers in thecabazitaxel-albumin nanoparticle composition comprising argininehydrochloride obtained in Example 4 and in the cabazitaxel albuminnanoparticle composition without arginine hydrochloride obtained inComparative Example 5 were determined under the accelerated conditionsat 40° C. by using the same method as in Test Example 3. The testresults were shown in Table 4.

TABLE 5 Contents of albumin monomers, dimers and polymers in thecomposition of Example 4 and in the composition of Comparative Example 5at 40° C. Condi- Composition tion Example 4 Example 5 40° C. monomersdimers polymers monomers dimers polymers 0 93.4% 4.2% 1.2% 93.1%  4.1%1.4% month 1 92.8% 4.3% 1.5% 86.6%  7.0% 3.2% month 3 91.5% 4.6% 2.0%79.2% 10.4% 5.2% months 6 90.8% 4.9% 2.2% 74.6% 14.2% 5.6% months

In summary, the above results showed that, the addition of an amino acidhaving a relative molecular mass of 145-175 or a salt thereof to ananoparticle composition comprising a taxane compound could effectivelyinhibit the increase of albumin dimers and polymers and the degradationof albumin monomers in the composition, control the contents of albuminmonomers, dimers and polymers in the composition, and effectivelystabilize albumin monomers therein, and the albumin stabilizing effectthereof was superior to commonly used albumin stabilizers, keep albuminmonomer from being degraded substantially, to ensure the safety inclinical application.

In addition to those described herein, according to the abovedescriptions, various modifications of the invention will be obvious fora person skilled in the art. Such modifications also fall within thescope of the appended claims. Each of the references (including allpatents, patent applications, journal articles, books, and any otherpublications) cited in this application is hereby incorporated byreference in its entirety.

What is claimed is:
 1. A method for producing an albumin containingpharmaceutical composition wherein the formation of albumin dimersand/or the increase of albumin dimers during preparation, storage and/oruse of the pharmaceutical composition is inhibited, comprising addingduring the preparation of an albumin containing pharmaceuticalcomposition at least one amino acid having a relative molecular mass of145-175 or a salt thereof, wherein the amino acid or the salt thereofinhibits the formation and/or the increase of albumin dimers in thepharmaceutical composition during preparation, storage and/or usethereof.
 2. The method according to claim 1, wherein, the addition ofsaid at least one amino acid having a relative molecular mass of 145-175or salt thereof further inhibits the formation and/or the increase ofalbumin polymers during the preparation, storage and/or use of thepharmaceutical composition.
 3. (canceled)
 4. The method according toclaim 1, wherein, the amino acid is selected from one or more ofarginine, histidine and lysine.
 5. The method according to claim 1,wherein, the pharmaceutical composition comprises a taxane compound asactive ingredient.
 6. (canceled)
 7. The method according to claim 1,wherein, the amino acid or the salt thereof is the sole stabilizer addedduring the preparation of the pharmaceutical composition.
 8. A stablealbumin containing pharmaceutical composition, comprising (i) an albuminand (ii) at least one amino acid having a relative molecular mass of145-175 or a salt thereof, wherein the amount of albumin dimers presentin said pharmaceutical composition after the pharmaceutical compositionis stored for at least one year constitute no more than 10% of the totalamount of the albumins contained in the pharmaceutical composition. 9.The albumin containing pharmaceutical composition according to claim 8,wherein the amount of albumin dimers present in said pharmaceuticalcomposition after the pharmaceutical composition is stored for at leastone year constitute no more than 6% of the total amount of the albuminscontained in the pharmaceutical composition.
 10. The albumin containingpharmaceutical composition according to claim 8, wherein, the amino acidis selected from one or more of arginine, histidine and lysine.
 11. Thealbumin containing pharmaceutical composition according to claim 8,wherein, the pharmaceutical composition comprises a taxane compound asan active ingredient. 12-13. (canceled)
 14. A method for preparing thepharmaceutical composition according to claim 8, comprising thefollowing steps of: dissolving a taxane compound in a suitable organicsolvent to obtain a solution of the taxane compound; dissolving ordiluting an albumin in an aqueous solvent to obtain an aqueous solutionof the albumin; obtaining an aqueous solution of the amino acid having arelative molecular mass of 145-175 or the salt thereof using water forinjection; and combining the solution of the taxane compound, theaqueous solution of the albumin and the aqueous solution of the aminoacid having a relative molecular mass of 145-175 or the salt thereof bydispersive-mixing under high shear force, then ultrafiltration, andsterilization by filtration. 15-17. (canceled)
 18. The method accordingto claim 1, wherein the amount of albumin dimers present in thepharmaceutical composition is no more than 10% of the total amount ofthe albumins contained therein.
 19. The method according to claim 1,wherein the amount of albumin polymers present in the pharmaceuticalcomposition is no more than 6% of the total amount of the albuminscontained therein.
 20. The method according to claim 1, wherein theamino acid is arginine.
 21. The method according to claim 1, wherein theamino acid or the salt thereof and albumin are present in a weight ratioof from 0.1:1 to 10:1.
 22. The method according to claim 1, wherein theamino acid or the salt thereof and albumin are present in a weight ratioof from 0.2:1 to 5:1.
 23. The method according to claim 5, wherein thetaxane compound is selected from the group consisting of paclitaxel,docetaxel, cabazitaxel, and derivatives thereof.
 24. The albuminpharmaceutical composition according to claim 8, wherein the amino acidis arginine.
 25. The albumin containing pharmaceutical compositionaccording to claim 8, wherein the amino acid or the salt thereof andalbumin are present in a weight ratio ranging from 0.1:1 to 10:1. 26.The albumin containing pharmaceutical composition according to claim 8,wherein the amino acid or the salt thereof and albumin are present in aweight ratio ranging from 0.2:1 to 5:1.
 27. The albumin containingpharmaceutical composition according to claim 11, wherein the taxanecompound is selected from the group consisting of paclitaxel, docetaxel,cabazitaxel, and derivatives thereof.
 28. A method for treating acancer, comprising administering to a subject in need a therapeuticallyeffective amount of a pharmaceutical composition according to claim 8.29. The method according to claim 28, wherein the cancer is selectedfrom one or more of prostate cancer, colon cancer, breast cancer, headand neck cancer, pancreatic cancer, lung cancer, ovarian cancer,multiple myeloma, renal cell carcinoma, melanoma, liver cancer, gastriccancer, and kidney cancer.
 30. The method according to claim 24, furthercomprising a step of lyophilization after the sterilization byfiltration, wherein the method does not comprise a step of adding alyoprotectant prior to the lyophilization, wherein the lyoprotectantcomprises one or more of trehalose, sucrose, maltose, lactose andglycine.